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Endorepellin-evoked Autophagy Contributes to Angiostasis*

  • Atul Goyal
    Footnotes
    Affiliations
    From the Department of Pathology, Anatomy, and Cell Biology and the Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and
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  • Maria A. Gubbiotti
    Footnotes
    Affiliations
    From the Department of Pathology, Anatomy, and Cell Biology and the Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and
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  • Daphney R. Chery
    Footnotes
    Affiliations
    the School of Biomedical Engineering, Science, and Health Systems, Drexel University, Philadelphia, Pennsylvania 19104
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  • Lin Han
    Footnotes
    Affiliations
    the School of Biomedical Engineering, Science, and Health Systems, Drexel University, Philadelphia, Pennsylvania 19104
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  • Renato V. Iozzo
    Correspondence
    To whom correspondence should be addressed: Dept. of Pathology, Anatomy, and Cell Biology, 1020 Locust St., Ste. 336 JAH, Philadelphia, PA 19107. Tel.: 215-503-2208; Fax: 215-923-7969;
    Affiliations
    From the Department of Pathology, Anatomy, and Cell Biology and the Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grants RO1 CA39481, RO1 CA47282, and RO1 CA164462 (to R. V. I.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    1 Both authors contributed equally to this work.
    2 Supported in part by National Institutes of Health Training Grant T32 AA07463.
    3 Supported by the Department of Education GAANN Program and a Drexel Interdisciplinary Collaboration and Research Enterprise (iCARE) for Healthcare fellowship.
    4 Supported by a Drexel faculty startup grant.
Open AccessPublished:July 19, 2016DOI:https://doi.org/10.1074/jbc.M116.740266
      Endorepellin, the C-terminal domain of perlecan, is an angiostatic molecule that acts as a potent inducer of autophagy via its interaction with VEGFR2. In this study, we examined the effect of endorepellin on endothelial cells using atomic force microscopy. Soluble endorepellin caused morphological and biophysical changes such as an increase in cell surface roughness and cell height. Surprisingly, these changes were not accompanied by alterations in the endothelial cell elastic modulus. We discovered that endorepellin-induced autophagic flux led to co-localization of mammalian target of rapamycin with LC3-positive autophagosomes. Endorepellin functioned upstream of AMP-activated kinase α, as compound C, an inhibitor of AMP-activated kinase α, abrogated endorepellin-mediated activation and co-localization of Beclin 1 and LC3, thereby reducing autophagic progression. Functionally, we discovered that both endorepellin and Torin 1, a canonical autophagic inducer, blunted ex vivo angiogenesis. We conclude that autophagy is a novel mechanism by which endorepellin promotes angiostasis independent of nutrient deprivation.

      Introduction

      Perlecan is a large modular proteoglycan that belongs to the pericellular and basement membrane class of proteoglycans (
      • Iozzo R.V.
      Basement membrane proteoglycans: from cellar to ceiling.
      ,
      • Iozzo R.V.
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      Proteoglycan form and function: A comprehensive nomenclature of proteoglycans.
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      Border patrol: insights into the unique role of perlecan/heparan sulfate proteoglycan 2 at cell and tissue borders.
      ). Its protein core alone is ∼500 kDa and is comprised of five domains, with three heparan sulfate chains attached at the N terminus. It is encoded by an ∼115-kb gene, HSPG2 (
      • Noonan D.M.
      • Fulle A.
      • Valente P.
      • Cai S.
      • Horigan E.
      • Sasaki M.
      • Yamada Y.
      • Hassell J.R.
      The complete sequence of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive similarity with laminin A chain, low density lipoprotein-receptor, and the neural cell adhesion molecule.
      ,
      • Murdoch A.D.
      • Dodge G.R.
      • Cohen I.
      • Tuan R.S.
      • Iozzo R.V.
      Primary structure of the human heparan sulfate proteoglycan from basement membrane (HSPG2/perlecan): a chimeric molecule with multiple domains homologous to the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor.
      ), and is expressed in a variety of tissues, both vascular and avascular (
      • Carson D.D.
      • Tang J.-P.
      • Julian J.
      Heparan sulfate proteoglycan (perlecan) expression by mouse embryos during acquisition of attachment competence.
      ,
      • Handler M.
      • Yurchenco P.D.
      • Iozzo R.V.
      Developmental expression of perlecan during murine embryogenesis.
      ). The biological functions of perlecan span a range of processes, including cell adhesion (
      • Whitelock J.M.
      • Graham L.D.
      • Melrose J.
      • Murdoch A.D.
      • Iozzo R.V.
      • Underwood P.A.
      Human perlecan immunopurified from different endothelial cell sources has different adhesive properties for vascular cells.
      ,
      • Lord M.S.
      • Chuang C.Y.
      • Melrose J.
      • Davies M.J.
      • Iozzo R.V.
      • Whitelock J.M.
      The role of vascular-derived perlecan in modulating cell adhesion, proliferation and growth factor signaling.
      ), endocytosis (
      • Christianson H.C.
      • Belting M.
      Heparan sulfate proteoglycan as a cell-surface endocytosis receptor.
      ), bone and cartilage formation (
      • Jochmann K.
      • Bachvarova V.
      • Vortkamp A.
      Heparan sulfate as a regulator of endochondral ossification and osteochondroma development.
      ,
      • Wilusz R.E.
      • Sanchez-Adams J.
      • Guilak F.
      The structure and function of the pericellular matrix of articular cartilage.
      ), inflammation and wound healing (
      • Lord M.S.
      • Jung M.
      • Cheng B.
      • Whitelock J.M.
      Transcriptional complexity of the HSPG2 gene in the human mast cell line, HMC-1.
      ,
      • Jung M.
      • Lord M.S.
      • Cheng B.
      • Lyons J.G.
      • Alkhouri H.
      • Hughes J.M.
      • McCarthy S.J.
      • Iozzo R.V.
      • Whitelock J.M.
      Mast cells produce novel shorter forms of perlecan that contain functional endorepellin: a role in angiogenesis and wound healing.
      ), thrombosis (
      • Nugent M.A.
      • Nugent H.M.
      • Iozzo R.V.
      • Sanchack K.
      • Edelman E.R.
      Perlecan is required to inhibit thrombosis after deep vascular injury and contributes to endothelial cell-mediated inhibition of intimal hyperplasia.
      ), lipid metabolism (
      • Fuki I.V.
      • Iozzo R.V.
      • Williams K.J.
      Perlecan heparan sulfate proteoglycan: a novel receptor that mediates a distinct pathway for ligand catabolism.
      ), autophagy (
      • Ning L.
      • Xu Z.
      • Furuya N.
      • Nonaka R.
      • Yamada Y.
      • Arikawa-Hirasawa E.
      Perlecan inhibits autophagy to maintain muscle homeostasis in mouse soleus muscle.
      ), tumor angiogenesis and invasiveness (
      • Cohen I.R.
      • Murdoch A.D.
      • Naso M.F.
      • Marchetti D.
      • Berd D.
      • Iozzo R.V.
      Abnormal expression of perlecan proteoglycan in metastatic melanomas.
      • Mathiak M.
      • Yenisey C.
      • Grant D.S.
      • Sharma B.
      • Iozzo R.V.
      A role for perlecan in the suppression of growth and invasion in fibrosarcoma cells.
      ,
      • Iozzo R.V.
      • Zoeller J.J.
      • Nyström A.
      Basement membrane proteoglycans: modulators par excellence of cancer growth and angiogenesis.
      ,
      • Iozzo R.V.
      • Sanderson R.D.
      Proteoglycans in cancer biology, tumour microenvironment and angiogenesis.
      ,
      • Grindel B.J.
      • Martinez J.R.
      • Pennington C.L.
      • Muldoon M.
      • Stave J.
      • Chung L.W.
      • Farach-Carson M.C.
      Matrilysin/matrix metalloproteinase-7(MMP7) cleavage of perlecan/HSPG2 creates a molecular switch to alter prostate cancer cell behavior.
      • Poluzzi C.
      • Iozzo R.V.
      • Schaefer L.
      Endostatin and endorepellin: a common route of action for similar angiostatic cancer avengers.
      ), and cardiovascular development (
      • Zoeller J.J.
      • McQuillan A.
      • Whitelock J.
      • Ho S.-Y.
      • Iozzo R.V.
      A central function for perlecan in skeletal muscle and cardiovascular development.
      ), where its angiogenic properties are among the most interesting. Perlecan transcription is also induced by TGF-β (
      • Iozzo R.V.
      • Pillarisetti J.
      • Sharma B.
      • Murdoch A.D.
      • Danielson K.G.
      • Uitto J.
      • Mauviel A.
      Structural and functional characterization of the human perlecan gene promoter: transcriptional activation by transforming factor-β via a nuclear factor 1-binding element.
      ) and is rapidly repressed by interferon γ (
      • Sharma B.
      • Iozzo R.V.
      Transcriptional silencing of perlecan gene expression by interferon-γ.
      ).
      The network of heparan sulfate/growth factor interactions is a key regulator of angiogenesis (
      • Peysselon F.
      • Ricard-Blum S.
      Heparin-protein interactions: from affinity and kinetics to biological roles: application to an interaction network regulating angiogenesis.
      ). Perlecan sequesters VEGFA and FGFs via its N-terminal heparan sulfate side chains, which are then released by heparanases and subsequently bind to their cognate receptors, resulting in the induction of angiogenesis (
      • Lord M.S.
      • Chuang C.Y.
      • Melrose J.
      • Davies M.J.
      • Iozzo R.V.
      • Whitelock J.M.
      The role of vascular-derived perlecan in modulating cell adhesion, proliferation and growth factor signaling.
      ,
      • Iozzo R.V.
      • San Antonio J.D.
      Heparan sulfate proteoglycans: heavy hitters in the angiogenesis arena.
      ,
      • Whitelock J.M.
      • Melrose J.
      • Iozzo R.V.
      Diverse cell signaling events modulated by perlecan.
      ). In addition, there is a feedforward loop in that VEGFA induces perlecan synthesis via the activation of VEGFR2, leading to increased angiogenesis (
      • Kaji T.
      • Yamamoto C.
      • Oh-i M.
      • Fujiwara Y.
      • Yamazaki Y.
      • Morita T.
      • Plaas A.H.
      • Wight T.N.
      The vascular endothelial growth factor VEGF165 induces perlecan synthesis via VEGF receptor-2 in cultured human brain microvascular endothelial cells.
      ,
      • Neill T.
      • Schaefer L.
      • Iozzo R.V.
      Instructive roles of extracellular matrix on autophagy.
      ). Indeed, antisense targeting of perlecan inhibits in vivo tumor angiogenesis (
      • Sharma B.
      • Handler M.
      • Eichstetter I.
      • Whitelock J.M.
      • Nugent M.A.
      • Iozzo R.V.
      Antisense targeting of perlecan blocks tumor growth and angiogenesis in vivo.
      ). During development, perlecan acts as a scaffold for blood vessel formation, and a restriction of Hspg2 expression in early embryogenesis results in cardiovascular defects (
      • Handler M.
      • Yurchenco P.D.
      • Iozzo R.V.
      Developmental expression of perlecan during murine embryogenesis.
      ,
      • Gustafsson E.
      • Almonte-Becerril M.
      • Bloch W.
      • Costell M.
      Perlecan maintains microvessel integrity in vivo and modulates their formation in vitro.
      ).
      In contrast, the C-terminal domain V of perlecan, known as endorepellin, exhibits angiostatic properties (
      • Mongiat M.
      • Sweeney S.M.
      • San Antonio J.D.
      • Fu J.
      • Iozzo R.V.
      Endorepellin, a novel inhibitor of angiogenesis derived from the C terminus of perlecan.
      ). Endorepellin is found in vivo, where it is proteolytically processed from perlecan (
      • Jung M.
      • Lord M.S.
      • Cheng B.
      • Lyons J.G.
      • Alkhouri H.
      • Hughes J.M.
      • McCarthy S.J.
      • Iozzo R.V.
      • Whitelock J.M.
      Mast cells produce novel shorter forms of perlecan that contain functional endorepellin: a role in angiogenesis and wound healing.
      ) via matrix metalloproteinases, a family of enzymes involved in a multitude of biological processes (
      • Arpino V.
      • Brock M.
      • Gill S.E.
      The role of TIMPs in regulation of extracellular matrix proteolysis.
      • Wells J.M.
      • Gaggar A.
      • Blalock J.E.
      MMP generated matrikines.
      ,
      • Deryugina E.I.
      • Quigley J.P.
      Tumor angiogenesis: MMP-mediated induction of intravasation- and metastasis-sustaining neovasculature.
      ,
      • Rohani M.G.
      • Parks W.C.
      Matrix remodeling by MMPs during wound repair.
      • Duarte S.
      • Baber J.
      • Fujii T.
      • Coito A.J.
      Matrix metalloproteinases in liver injury, repair and fibrosis.
      ). Although perlecan mRNA can undergo alternative splicing, no evidence exists for endorepellin production in vivo via this mechanism (
      • Jung M.
      • Lord M.S.
      • Cheng B.
      • Lyons J.G.
      • Alkhouri H.
      • Hughes J.M.
      • McCarthy S.J.
      • Iozzo R.V.
      • Whitelock J.M.
      Mast cells produce novel shorter forms of perlecan that contain functional endorepellin: a role in angiogenesis and wound healing.
      ). This domain of perlecan is an 85-kDa protein comprised of four EGF-like repeats and three laminin-like globular domains (LG1–3). Structurally, LG2/LG3 domains of endorepellin are separated by two EGF-like repeats that can be cleaved by BMP1/Tolloid-like proteases (
      • Vadon-Le Goff S.
      • Hulmes D.J.
      • Moali C.
      BMP-1/tolloid-like proteinases synchronize matrix assembly with growth factor activation to promote morphogenesis and tissue remodeling.
      ,
      • Apte S.S.
      • Parks W.C.
      Metalloproteinases: A parade of functions in matrix biology and an outlook for the future.
      ) to release the LG3 domain (
      • Gonzalez E.M.
      • Reed C.C.
      • Bix G.
      • Fu J.
      • Zhang Y.
      • Gopalakrishnan B.
      • Greenspan D.S.
      • Iozzo R.V.
      BMP-1/Tolloid-like metalloproteases process endorepellin, the angiostatic C-terminal fragment of perlecan.
      ).
      As its name implies, endorepellin is an inhibitor of endothelial cell migration and capillary morphogenesis, thus preventing the formation of new blood vessels (
      • Mongiat M.
      • Sweeney S.M.
      • San Antonio J.D.
      • Fu J.
      • Iozzo R.V.
      Endorepellin, a novel inhibitor of angiogenesis derived from the C terminus of perlecan.
      ). These functional properties result from a “dual receptor antagonism” through its binding to VEGFR2 and α2β1 integrin (
      • Goyal A.
      • Pal N.
      • Concannon M.
      • Paul M.
      • Doran M.
      • Poluzzi C.
      • Sekiguchi K.
      • Whitelock J.M.
      • Neill T.
      • Iozzo R.V.
      Endorepellin, the angiostatic module of perlecan, interacts with both the α2β1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2).
      ): LG1/2 bind to the IgG3–5 repeats in the VEGFR2 ectodomain, whereas LG3 binds to α2β1 integrin (
      • Willis C.D.
      • Poluzzi C.
      • Mongiat M.
      • Iozzo R.V.
      Endorepellin laminin-like globular repeat 1/2 domains bind Ig3–5 of vascular endothelial growth factor (VEGF) receptor 2 and block pro-angiogenic signaling by VEGFA in endothelial cells.
      ). This biological interaction leads to rapid internalization of both receptors and, ultimately, attenuation of the PI3K/phosphoinositide-dependent kinase/Akt/mTOR
      The abbreviations used are: mTOR,mammalian target of rapamycin; AFM,atomic force microscopy; HUVEC,human umbilical vein endothelial cell(s); HBSS,Hanks' balanced salt solution; AMPK,AMP kinase.;
      and PKC/JNK/AP1 pathways and a decrease in expression of VEGFA, thus contributing to the anti-angiogenic activity of endorepellin (
      • Goyal A.
      • Poluzzi C.
      • Willis C.D.
      • Smythies J.
      • Shellard A.
      • Neill T.
      • Iozzo R.V.
      Endorepellin affects angiogenesis by antagonizing diverse VEGFR2- evoked signaling pathways: transcriptional repression of HIF-1α and VEGFA and concurrent inhibition of NFAT1 activation.
      ).
      In vivo studies have shown that endorepellin specifically targets the tumor vasculature and inhibits tumor angiogenesis (
      • Bix G.
      • Castello R.
      • Burrows M.
      • Zoeller J.J.
      • Weech M.
      • Iozzo R.A.
      • Cardi C.
      • Thakur M.L.
      • Barker C.A.
      • Camphausen K.
      • Iozzo R.V.
      Endorepellin in vivo: targeting the tumor vasculature and retarding cancer growth and metabolism.
      ). This bioactivity leads to inhibition of tumor growth without inducing apoptosis. Recently, we have discovered that soluble endorepellin induces autophagy in endothelial cells via the binding of its LG1/2 domains to VEGFR2 (
      • Poluzzi C.
      • Casulli J.
      • Goyal A.
      • Mercer T.J.
      • Neill T.
      • Iozzo R.V.
      Endorepellin evokes autophagy in endothelial cells.
      ). This process occurs independently of the α2β1 integrin and induces several autophagic markers (Beclin 1, LC3, and p62) under nutrient-enriched conditions (
      • Poluzzi C.
      • Casulli J.
      • Goyal A.
      • Mercer T.J.
      • Neill T.
      • Iozzo R.V.
      Endorepellin evokes autophagy in endothelial cells.
      ). In this study, we examined in detail the physical properties of endothelial cells treated with endorepellin via atomic force microscopy (AFM) imaging and nanoindentation. We further elucidated the mechanism behind endorepellin-evoked autophagy. Specifically, we found that endorepellin evoked phosphorylation of AMPKα at Thr172, a key residue necessary for autophagic progression. Moreover, endorepellin blunted vessel sprouting in ex vivo angiogenesis assays, and this bioactivity was blocked by halting AMPKα activation. Thus, we propose a new mechanism by which a fragment of an extracellular proteoglycan links angiostasis to autophagy.

      Results

      Endorepellin and Torin 1 Evoke Nanoscale Molecular Bumps in Endothelial Cells

      To determine the nanoscale structural changes in porcine (PAER2) cells and human endothelial cells (HUVEC) evoked by endorepellin or Torin 1, a selective inhibitor of mTOR (
      • Thoreen C.C.
      • Kang S.A.
      • Chang J.W.
      • Liu Q.
      • Zhang J.
      • Gao Y.
      • Reichling L.J.
      • Sim T.
      • Sabatini D.M.
      • Gray N.S.
      An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1.
      ), we utilized tapping mode AFM imaging, which quantifies cell surface topography at nanoscale spatial resolution. We discovered that, although the vehicle-treated PAER2 cell surface was relatively smooth (Fig. 1A), that of endorepellin-treated cells (Fig. 1B) revealed increased surface roughness with the formation of discrete bumps. Identical bumps were detected in the cells treated with Torin 1 (Fig. 1C).
      Figure thumbnail gr1
      FIGURE 1Endorepellin induces nanoscale molecular changes in porcine endothelial cells overexpressing VEGFR2. A–F, representative AFM images of PAER2 cells treated with vehicle, endorepellin (200 nm, 6 h), and Torin 1 (20 nm, 2 h). Three independent experiments, also serving as biological replicates, of each condition were performed with a minimum analysis of at least 5 cells/condition/experiment (technical replicates). The white arrows indicate the molecular bumps. A–C, three-dimensional images of the entire cell. D–F, magnified two-dimensional images (40 μm) of the cells. The brown-scale colors in the images indicate different heights, with light and dark colors corresponding to higher and lower topography, respectively (see the associated scale bar). G–I, line-scanned profiles of the blue and red lines in the magnified images shown in D–F.
      Next we determined the height of these bumps from the three-dimensional images (Fig. 1, D–F). Line scanning profiles of the three-dimensional bumps showed marked elevation in both endorepellin-treated (Fig. 1H) and Torin 1-treated cells (Fig. 1I), suggesting that these elevations may represent autophagosomes. In comparison, vehicle-treated cells exhibited a uniform height (Fig. 1G).
      As the PAER2 cells, the human counterparts showed identical formation of nanomolecular bumps on their cell surface (Fig. 2, E–G) compared with vehicle-treated HUVEC (Fig. 2, A–C). Line scanning profiles of the three-dimensional bumps showed marked elevation in HUVEC treated with endorepellin (Fig. 2H) vis à vis cells treated with vehicle (Fig. 2D). We also used low-magnification AFM images (Fig. 2, I–K) to quantify the number of these bumps, which we interpret as autophagosomes. There was a significant increase in the number of autophagosome-like structures evoked by either endorepellin or Torin 1 vis à vis vehicle-treated cells (p < 0.001, Fig. 2L).
      Figure thumbnail gr2
      FIGURE 2Human umbilical vein endothelial cells exhibit biophysical changes in response to endorepellin treatment. A–C, representative AFM images of HUVEC treated with vehicle. E–G, representative AFM images of HUVEC treated with endorepellin (200 nm, 6 h). A and E and B and F, full and magnified three-dimensional images of the cell, respectively. C and G, two-dimensional magnified images. D and H, line-scanned profiles of the blue and red lines in the magnified images shown in C and G. I–K, low-magnification AFM images of HUVEC representing treatment with vehicle, endorepellin (200 nm, 6 h), and Torin 1 (20 nm, 2 h). Note the presence of increased discrete elevated points in the endorepellin- and Torin 1-treated cells, as represented by the white circular areas. These points are hypothesized to be autophagosomes. L, quantification of the number of autophagosome-like structures per cell in the images in I–K. M, surface roughness parameter, Ra, as a function of endorepellin or Torin 1 treatment in HUVEC. For quantification: Vehicle, 72 cells from three biological replicates obtained from three independent experiments; Endorepellin, 50 cells from three biological replicates obtained from three independent experiments; Torin 1, 48 cells from three biological replicates obtained from three independent experiments. *, p < 0.05; ***, p < 0.001; Student's t test.
      To quantify the changes in cell morphology associated with autophagy, we analyzed cell surface roughness. The two surface roughness parameters, Ra (arithmetic mean height) and Rq (root mean square height), were determined in an area of ∼36 μm2. We observed that, upon induction of autophagosome-like structures, the cell surface roughness (Ra) was markedly increased in both endorepellin-treated (p < 0.05, Fig. 2M) and Torin 1-treated HUVEC (p < 0.001, Fig. 2M). Rq values were similar to the Ra values (data not shown). We interpret these findings as representative of autophagosome generation within the cytoplasm of endothelial cells derived from both porcine and human large vessels.

      Despite Cell Surface Nanostructural Changes, Endorepellin Does Not Alter the Elastic Modulus of Endothelial Cells

      Following this observation of nanostructural changes in the endorepellin-treated endothelial cells, we hypothesized that autophagosome formation, induced both by endorepellin and nutrient deprivation, would result in changes in the cellular elastic modulus. To this end, we utilized a special type of AFM where nanoindentation was performed by a microspherical tip that directly indents the cell surface while the cells are alive and grown in appropriate culture medium (
      • Mathur A.B.
      • Truskey G.A.
      • Reichert W.M.
      Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells.
      ,
      • Lekka M.
      Discrimination between normal and cancerous cells using AFM.
      ). This configuration provides information regarding the dynamic biomechanical properties of the target cells in response to different cellular milieus, allowing for an accurate representation of the changes in cellular elasticity under varying conditions. Although the focus of this study was on endorepellin-induced biomechanical changes, we also utilized Hanks' balanced salt solution (HBSS), a nutrient deprivation method used to induce autophagy through inhibition of mTOR signaling (
      • Levine B.
      • Kroemer G.
      Autophagy in the pathogenesis of disease.
      ). This acted as a positive control for autophagic induction to determine whether autophagy itself could alter the elastic modulus of endothelial cells.
      We surmised that the presence of autophagosomes would increase the stiffness of the cells. To our great surprise, despite the presence of structural differences visible by differential interference contrast microscopy in both the endorepellin- and HBSS-treated cells vis à vis vehicle-treated cells (Fig. 3, A–D), the elastic modulus of these HUVEC was not significantly altered (Fig. 3E). Intriguingly, the positive control cells treated with HBSS also did not show any significant change in elastic modulus, suggesting that the formation of autophagosomes may not result in changes in stiffness as measured at the cell surface, at least in endothelial cells.
      Figure thumbnail gr3
      FIGURE 3Endorepellin and autophagy do not significantly alter the elastic modulus of endothelial cells. A–D, differential interference contrast microscopy images of HUVEC treated with vehicle, endorepellin, or HBSS. Note the presence of autophagosome-like structures, as depicted by the white arrows in the endorepellin- and HBSS-treated cells. E, measurement of the elastic modulus of HUVEC treated with conditions in A–D determined via AFM nanoindentation. For quantification: Vehicle, n = 103 technical replicates from six biological replicates obtained from six independent experiments, Endorepellin, 200 nm endorepellin for 6 h, n = 126 technical replicates from eight biological replicates obtained from eight independent experiments; HBSS, 4 h, n = 71 technical replicates from four biological replicates obtained from four independent experiments. The p values were obtained using Student's t test.

      Endorepellin Evokes Autophagic Flux and Co-localization of mTOR and LC3 into Puncta

      Following this biophysical analysis, we sought to delve deeper into the mechanism by which endorepellin contributes to the induction and/or progression of the autophagic process. First, we validated our cell system by performing autophagic flux experiments. To this end, HUVEC were treated with or without chloroquine, an inhibitor of the fusion of the lysosome with the autophagosome, which allows for the build-up of autophagic intermediates (
      • Levine B.
      • Kroemer G.
      Autophagy in the pathogenesis of disease.
      ). Relative levels of LC3-II were measured via immunoblotting to ascertain autophagic activity. We found an increase in LC3-II upon addition of endorepellin with chloroquine in comparison with chloroquine alone (Fig. 4, A and B), thereby validating induction of autophagy by soluble endorepellin.
      Figure thumbnail gr4
      FIGURE 4Endorepellin induces autophagic flux and mobilization of mTOR into LC3-positive autophagosomes. A, representative immunoblotting of HUVEC lysates following endorepellin treatment in the absence and presence of chloroquine. B, quantification of LC3-II using one technical replicate from each of six biological samples obtained from six independent experiments as seen in A, normalized to GAPDH. C–E, mTOR and LC3 staining in HUVEC following treatment with vehicle, endorepellin (200 nm, 6 h), or Torin 1 (20 nm, 2 h). Scale bar = 10 μm. The white arrows indicate autophagosomes. F, quantification of the number of autophagosomes (LC3- and mTOR-positive) per cell in HUVEC treated with vehicle (n = 76 cells from three biological replicates obtained from three separate experiments), endorepellin (n = 49 cells from three biological replicates obtained from three separate experiments), or Torin 1 (n = 60 cells from three biological replicates obtained from three separate experiments). *, p < 0.05, Student's t test.
      Next we determined the effects on the endorepellin-evoked redistribution of mTOR, a previously reported target of endorepellin (
      • Goyal A.
      • Poluzzi C.
      • Willis C.D.
      • Smythies J.
      • Shellard A.
      • Neill T.
      • Iozzo R.V.
      Endorepellin affects angiogenesis by antagonizing diverse VEGFR2- evoked signaling pathways: transcriptional repression of HIF-1α and VEGFA and concurrent inhibition of NFAT1 activation.
      ). Under basal conditions, most of the LC3-positive puncta were not associated with mTOR (Fig. 4C). However, upon endorepellin treatment, the majority of LC3 immunoreactivity co-distributed with mTOR into large, autophagosome-like structures (Fig. 4D) in a fashion similar to Torin 1-treated HUVEC (Fig. 4E). Quantification of three independent experiments showed a significant increase in the number of mTOR/LC3-positive autophagosomes per cell upon treatment with endorepellin (p < 0.05, Fig. 4F) or Torin 1 (p < 0.05, Fig. 4F) vis à vis vehicle-treated cells. Collectively, these findings corroborate the nanoscale molecular changes described above and suggest that the pro-autophagic activity of endorepellin can further evoke down-regulation of mTOR via autophagic clearance.

      Endorepellin Promotes Autophagy via AMPKα Activation

      It is well established that AMPK plays a key function in regulating autophagy and mTOR activity following phosphorylation of its catalytic α subunit at Thr172 (
      • Kuhajda F.P.
      AMP-activated protein kinase and human cancer: cancer metabolism revisited.
      ,
      • Kim J.
      • Kundu M.
      • Viollet B.
      • Guan K.-L.
      AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1.
      ). We hypothesized that endorepellin could induce autophagy through a canonical activation of AMPKα. Under nutrient-rich conditions, endorepellin increased the phosphorylation of AMPKα at Thr172 over time (Fig. 5A), reaching a peak at 4 h, with levels almost three times greater than those of untreated cells (Fig. 5B).
      Figure thumbnail gr5
      FIGURE 5Endorepellin activates the autophagic pathway via AMPKα phosphorylation. A, representative immunoblots of P-AMPKα (Thr172) in response to endorepellin (200 nm) at different time points. B, quantification of P-AMPKα:AMPKα as shown in A. One-way analysis of variance was used for statistical analysis. C, representative immunoblots of P-AMPKα, total AMPKα, Beclin 1, and LC3-II following incubation with endorepellin (200 nm, 6 h), compound C (30 μm, 30 min pretreatment + 6 h), or endorepellin and compound C. D, quantification of the P-AMPKα:AMPKα ratio shown in C. E and F, quantification of Beclin 1 and LC3-II as shown in C after normalization to GAPDH. Quantification was performed using one technical replicate each from three biological replicates obtained from three independent experiments. D–F, *, p < 0.05, Student's t test.
      To further verify the role of AMPKα in endorepellin-induced autophagy, we treated HUVEC with either endorepellin and/or compound C, a potent inhibitor of AMPK (
      • Kim J.
      • Kundu M.
      • Viollet B.
      • Guan K.-L.
      AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1.
      ). Upon addition of this inhibitor, the phosphorylation of AMPKα was no longer detected in endorepellin-treated cells (Fig. 5, C and D). Compound C also attenuated the effect of endorepellin on LC3-II and Beclin 1 levels (Fig. 5, C, E, and F). These results indicate that the activation of AMPKα is involved in the endorepellin-mediated increase in the autophagic markers Beclin 1 and LC3-II.
      Next we investigated the role of AMPKα in endorepellin-mediated autophagy by imaging the intracellular movement of LC3 and Beclin 1 after treatment with compound C, endorepellin, or both. Endorepellin alone promoted the co-localization of Beclin 1 and LC3 into large autophagosomes compared with vehicle-treated cells (Fig. 6, A and B). In contrast, compound C significantly decreased the formation of endorepellin-induced Beclin 1/LC3-positive autophagosomes (Fig. 6D) relative to endorepellin alone (Fig. 6E).
      Figure thumbnail gr6
      FIGURE 6AMPKα and VEGFR2 are required for endorepellin-mediated autophagy in HUVEC. A–D, immunofluorescence images depicting the co-localization of Beclin 1 (green) and LC3-II (red) shown upon treatment with vehicle (n = 31 cells from three biological replicates obtained from three independent experiments), 200 nm endorepellin for 6 h (n = 36 cells from three biological replicates obtained from three independent experiments), 30 μm compound C for 30-min pretreatment + 6 h (n = 28 cells from three biological replicates obtained from three independent experiments), or compound C + endorepellin (n = 28 cells from three biological replicates obtained from three independent experiments) in HUVEC. Nuclei are stained with DAPI (blue). Scale bar = 10 μm. White arrows indicate autophagosomes. E, quantification of the number of autophagosomes per cell in HUVEC treated according to the conditions shown in A–D. F, representative immunoblots of HUVEC pretreated with scrambled siRNA (SiScr) or siRNA targeting VEGFR2 (siVEGFR2) following treatment with endorepellin (200 nm, 6 h). G–H, quantification of VEGFR2 after normalization to GAPDH (one technical replicate from each of three biological replicates obtained from three independent experiments) and P-AMPKα:AMPKα ratio (one technical replicate from each of three biological replicates obtained from three independent experiments) shown in F. *, p < 0.05; **, p < 0.01; ***, p < 0.001; Student's t test.

      VEGFR2 Is Required for Endorepellin-evoked Phosphorylation of AMPKα Thr172

      Next we investigated the relationship between AMPKα and VEGFR2 following treatment of endothelial cells with endorepellin by genetically targeting this receptor via RNAi. Following verification of successful knockdown of VEGFR2 (Fig. 6, F and G), we found that an ∼50% reduction in VEGFR2 protein levels prevented the endorepellin-evoked phosphorylation of AMPKα at Thr172 (Fig. 6F), and these data were significant vis à vis scrambled siRNA in three independent experiments (p < 0.01, Fig. 6H). We conclude that VEGFR2 is required for the proper activation of AMPKα following interaction at the cell surface with the LG1/2 domains of endorepellin (
      • Willis C.D.
      • Poluzzi C.
      • Mongiat M.
      • Iozzo R.V.
      Endorepellin laminin-like globular repeat 1/2 domains bind Ig3–5 of vascular endothelial growth factor (VEGF) receptor 2 and block pro-angiogenic signaling by VEGFA in endothelial cells.
      ), as its knockdown leads to a significant attenuation of endorepellin-induced autophagy.

      Endorepellin and Torin 1 Inhibit ex Vivo Angiogenesis

      Given its well established angiostatic activity (
      • Mongiat M.
      • Sweeney S.M.
      • San Antonio J.D.
      • Fu J.
      • Iozzo R.V.
      Endorepellin, a novel inhibitor of angiogenesis derived from the C terminus of perlecan.
      ,
      • Goyal A.
      • Pal N.
      • Concannon M.
      • Paul M.
      • Doran M.
      • Poluzzi C.
      • Sekiguchi K.
      • Whitelock J.M.
      • Neill T.
      • Iozzo R.V.
      Endorepellin, the angiostatic module of perlecan, interacts with both the α2β1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2).
      ,
      • Poluzzi C.
      • Casulli J.
      • Goyal A.
      • Mercer T.J.
      • Neill T.
      • Iozzo R.V.
      Endorepellin evokes autophagy in endothelial cells.
      ), we hypothesized that endorepellin could evoke autophagy and inhibit angiogenesis via a common pathway. To this end, we utilized an ex vivo model, the aortic ring assay (
      • Brill A.
      • Elinav H.
      • Varon D.
      Differential role of platelet granular mediators in angiogenesis.
      ). This assay closely mimics the in vivo environment required for angiogenesis, as it includes both endothelial and supporting cells, which surround the aorta in vivo (
      • Auerbach R.
      • Lewis R.
      • Shinners B.
      • Kubai L.
      • Akhtar N.
      Angiogenesis assays: a critical overview.
      ). In rings grown in a nutrient-rich environment, we observed a well structured microvessel network with clearly defined tubules and regular branching (Fig. 7, A and B). In contrast, the samples treated with endorepellin, using the same concentration (200 nm) as in the biochemical assays, showed a significant decrease in the number of microvessels growing from the rings (Fig. 7, C and D).
      Figure thumbnail gr7
      FIGURE 7Endorepellin and Torin 1 inhibit angiogenesis ex vivo in mouse aortic ring assays. A–D, light microscopy images depicting the difference in microvessel growth between vehicle-treated and endorepellin-treated rings (200 nm). B and D, close-up views of the sprouts in A and C. E–H, confocal images of vehicle-treated and endorepellin-treated rings (200 nm) where nuclei were stained with DAPI (blue) and endothelial cells were stained with CD31 (red). F and H, close-up views of sprouts found in E and G. I–L, representative confocal images of rings treated with either vehicle or Torin 1 (40 nm). Sprouts were stained with BS-1 Lectin (red), and nuclei were stained with DAPI (blue). K and L, magnified views of I and J. M, quantification of the number of sprouts in the vehicle-, endorepellin-, and Torin 1-treated rings. There were 16 technical and four biological replicates from four independent experiments for the first vehicle group, 18 technical and four biological replicates from four independent experiments for the endorepellin group, 11 technical and three biological replicates from three independent experiments for the second vehicle group, and 12 technical and three biological replicates from three independent experiments for the Torin 1 group. Scale bars = 100 μm. **, p < 0.01; Student's t test.
      To differentiate endothelial from non-endothelial cells, we immunostained the aortic ring explants with an antibody toward CD31/platelet endothelial cell adhesion molecule, a cell surface glycoprotein highly expressed by endothelial cells. The confocal images clearly showed that the sprouting vessel-like structures were indeed positive for CD31 (Fig. 7, E and F) and that endorepellin markedly reduced them (Fig. 7, G and H). Furthermore, treatment of aortic rings with Torin 1 revealed a pattern similar to that evoked by endorepellin (Fig. 7, I–L). Quantification of three to four independent experiments revealed a significant suppression of the number of sprouts per aortic ring by both endorepellin and Torin 1 vis à vis vehicle treatment (p < 0.01, Fig. 7M).

      Autophagic Inhibition Reverses the Angiostatic Response Triggered by Endorepellin

      Having established that both endorepellin and Torin 1 had comparable effects on angiogenesis, we sought to elucidate whether induction of autophagy was the impetus for the endorepellin-mediated angiostasis. To this end, we utilized compound C as a means to prevent endorepellin-mediated autophagy and angiostasis. In agreement with the findings presented above, endorepellin inhibited angiogenic sprouting from mouse aortic rings compared with those treated with vehicle (Fig. 8, A and B). Importantly, compound C blocked the inhibitory activity of endorepellin, as angiogenic sprouts grew to levels comparable with those seen in vehicle-treated rings (Fig. 8D). This outgrowth of vessels was statistically significant compared with endorepellin treatment alone but not significantly different from either vehicle-treated (Fig. 8A) or compound C-treated (Fig. 8C) rings (Fig. 8E). Thus, we can conclude that some of the anti-angiogenic effects of endorepellin are directly intertwined with its pro-autophagic capabilities, and, hence, we provide autophagy as a novel mechanism for the inhibition of neovascularization.
      Figure thumbnail gr8
      FIGURE 8Inhibition of autophagy by compound C reverses endorepellin-induced angiostasis. A–D, representative images of aortic rings treated with vehicle, 200 nm endorepellin, 1 μm compound C, or endorepellin and compound C. Vessels are indicated by the white arrows. Note the difference in visible sprouts between B and D. E, quantification of vessel number per ring for each of the treatment groups. There were 14 technical and six biological replicates from six independent experiments for the vehicle group, nine technical and four biological replicates from four independent experiments for the endorepellin group, eight technical and four biological replicates from four independent experiments for the compound C group, and 10 technical and four biological replicates from four independent experiments for the compound C and endorepellin group. Scale bars = 100 μm. *, p < 0.05; Student's t test.

      Discussion

      Autophagy is a catabolic process in which double or multiple membrane-delineated autophagosomes sequester cytoplasmic material and fuse with lysosomes, resulting in the degradation of their contents (
      • Choi A.M.
      • Ryter S.W.
      • Levine B.
      Autophagy in human health and disease.
      ). This process allows for the recycling of long-lived proteins and damaged organelles, thus maintaining cellular homeostasis. In addition to its function in the maintenance of cellular housekeeping under normal conditions, autophagy is also highly up-regulated during nutrient deprivation, hypoxia, and other unfavorable conditions, where it promotes cell survival (
      • Lum J.J.
      • Bauer D.E.
      • Kong M.
      • Harris M.H.
      • Li C.
      • Lindsten T.
      • Thompson C.B.
      Growth factor regulation of autophagy and cell survival in the absence of apoptosis.
      ). Given both the cytoprotective and sometimes cytotoxic functions of autophagy, defects in this process can contribute to a number of human pathologies, including cancer and neurodegenerative diseases (
      • Levine B.
      • Kroemer G.
      Autophagy in the pathogenesis of disease.
      ).
      Despite recent progress, the functional role of autophagy remains unclear in many biological contexts, particularly in angiogenesis, where its role remains controversial (
      • Du J.
      • Teng R.-J.
      • Guan T.
      • Eis A.
      • Kaul S.
      • Konduri G.G.
      • Shi Y.
      Role of autophagy in angiogenesis in aortic endothelial cells.
      ,
      • Kumar S.
      • Guru S.K.
      • Pathania A.S.
      • Kumar A.
      • Bhushan S.
      • Malik F.
      Autophagy triggered by magnolol derivative negatively regulates angiogenesis.
      ). Endorepellin, the C-terminal domain of perlecan, induces autophagy in endothelial cells (
      • Poluzzi C.
      • Casulli J.
      • Goyal A.
      • Mercer T.J.
      • Neill T.
      • Iozzo R.V.
      Endorepellin evokes autophagy in endothelial cells.
      ). Here we investigated in depth the mechanism through which endorepellin evokes autophagy and angiostasis and discovered that protracted autophagy under nutrient-rich conditions can negatively regulate angiogenesis.
      First, we analyzed the effect of endorepellin on the morphology and microphysical properties of endothelial cells using AFM. The latter enables high-resolution topographical imaging of a single cell surface with minimal sample preparation (
      • Braet F.
      • Seynaeve C.
      • De Zanger R.
      • Wisse E.
      Imaging surface and submembranous structures with the atomic force microscope: a study on living cancer cells, fibroblasts and macrophages.
      ). Notably, nanomolar concentrations of soluble endorepellin caused morphological and biophysical changes in endothelial cells derived from either porcine or human macrovessels, including an increase in cell surface roughness, cell height, and number of autophagosome-like structures. Similar changes were documented in cells treated with Torin 1, which induces autophagy through the inhibition of mTOR (
      • Thoreen C.C.
      • Kang S.A.
      • Chang J.W.
      • Liu Q.
      • Zhang J.
      • Gao Y.
      • Reichling L.J.
      • Sim T.
      • Sabatini D.M.
      • Gray N.S.
      An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1.
      ). Surprisingly, these changes were not accompanied by alterations in the endothelial cell elastic modulus. It is possible that endothelial cells do not incur any changes in elasticity following autophagic induction. However, it is also possible that, given the dynamic nature of autophagy, the lack of changes seen in the treated cells might be due to a high turnover rate of autophagosomes during this process. We note that there was slightly more variability in the modulus of endorepellin-treated cells compared with vehicle-treated samples, suggesting that there may be minor changes in modulus from transient formation of autophagosomes but that their rapid turnover makes any significant changes undetectable. Also, we must mention that, although the majority of cells undergo autophagy initiation following endorepellin treatment, it is possible that some cells do not. In these studies, the moduli were measured in cells chosen at random. Using AFM that can detect fluorescence-labeled autophagic markers could be a way to circumvent this limitation.
      We also wanted to scrutinize more thoroughly the intracellular signaling events that accompanied these biophysical changes observed at the cell surface. We focused on AMPKα and mTOR, two opposing regulators of the autophagic pathway (
      • Liang J.
      • Mills G.B.
      AMPK: A contextual oncogene or tumor suppressor?.
      ). When activated, AMPKα, the master nutrient-sensing enzyme, binds and phosphorylates ULK1, a key kinase in the initiation of this process (
      • Kim J.
      • Kundu M.
      • Viollet B.
      • Guan K.-L.
      AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1.
      ,
      • Lee J.W.
      • Park S.
      • Takahashi Y.
      • Wang H.-G.
      The association of AMPK with ULK1 regulates autophagy.
      ). Our working model (Fig. 9) delineates the necessity of phosphorylation of AMPKα at Thr172 for endorepellin-evoked autophagy. Interestingly, the kinetics of this phosphorylation are rather slow, peaking 4 h after endorepellin treatment, especially when compared with another extracellular matrix constituent, decorin (
      • Järveläinen H.
      • Sainio A.
      • Wight T.N.
      Pivotal role for decorin in angiogenesis.
      ,
      • Gubbiotti M.A.
      • Neill T.
      • Frey H.
      • Schaefer L.
      • Iozzo R.V.
      Decorin is an autophagy-inducible proteoglycan and is required for proper in vivo autophagy.
      • Gubbiotti M.A.
      • Iozzo R.V.
      Proteoglycans regulate autophagy via outside-in signaling: an emerging new concept.
      ). Decorin treatment also induces autophagy via AMPKα phosphorylation, which increases rapidly within 30 min of treatment and is maintained for up to 2 h (
      • Buraschi S.
      • Neill T.
      • Goyal A.
      • Poluzzi C.
      • Smythies J.
      • Owens R.T.
      • Schaefer L.
      • Torres A.
      • Iozzo R.V.
      Decorin causes autophagy in endothelial cells via Peg3.
      ,
      • Goyal A.
      • Neill T.
      • Owens R.T.
      • Schaefer L.
      • Iozzo R.V.
      Decorin activates AMPK, an energy sensor kinase, to induce autophagy in endothelial cells.
      ). However, the slow induction of AMPKα by endorepellin does correlate with increases in several autophagic proteins, including Beclin 1, LC3-II, Peg3, and p62 (
      • Poluzzi C.
      • Casulli J.
      • Goyal A.
      • Mercer T.J.
      • Neill T.
      • Iozzo R.V.
      Endorepellin evokes autophagy in endothelial cells.
      ).
      Figure thumbnail gr9
      FIGURE 9Working model depicting the interaction of endorepellin with VEGFR2 and subsequent downstream signaling events. The end result is a concurrent inhibition of angiogenesis and an induction of protracted autophagy. Both events can be efficiently blocked by suppressing VEGFR2 or by blocking AMPK enzymatic activity.
      Alongside the activation of the pro-autophagic pathway is the inhibition of the anti-autophagic mTOR pathway. mTOR also regulates ULK1 at different phosphorylation sites from AMPK, which results in deactivating ULK1 and, thus, inhibiting the initiation of autophagy. We have established previously that endorepellin attenuates the mTOR pathway via dephosphorylation at Ser2448 (
      • Goyal A.
      • Poluzzi C.
      • Willis C.D.
      • Smythies J.
      • Shellard A.
      • Neill T.
      • Iozzo R.V.
      Endorepellin affects angiogenesis by antagonizing diverse VEGFR2- evoked signaling pathways: transcriptional repression of HIF-1α and VEGFA and concurrent inhibition of NFAT1 activation.
      ). Here we found that, along with these biochemical changes, mTOR is taken up by autophagosomes upon endorepellin stimulation, in a fashion similar to that evoked by starvation in renal epithelial cells (
      • Yu L.
      • McPhee C.K.
      • Zheng L.
      • Mardones G.A.
      • Rong Y.
      • Peng J.
      • Mi N.
      • Zhao Y.
      • Liu Z.
      • Wan F.
      • Hailey D.W.
      • Oorschot V.
      • Klumperman J.
      • Baehrecke E.H.
      • Lenardo M.J.
      Termination of autophagy and reformation of lysosomes regulated by mTOR.
      ). We hypothesize that autophagosome-mediated degradation of mTOR caused by endorepellin/VEGFR2 signaling in endothelial cells would further enhance the pro-autophagic role of endorepellin. Proteoglycan receptors may thus play a role in regulating this important catabolic process (
      • Neill T.
      • Schaefer L.
      • Iozzo R.V.
      Decoding the matrix: instructive roles of proteoglycan receptors.
      ).
      Perhaps the most pivotal discovery of our study is the demonstration that autophagy can curtail neovascularization, as both endorepellin and Torin 1 reduce sprouting in ex vivo aortic ring assays. For the first time we were able to restore angiogenic sprouting following endorepellin treatment by blocking AMPKα-evoked autophagy using compound C. Interestingly, the synthesis of hyaluronan, a key component of the provisional angiogenic matrix under a complex metabolic control (
      • Vigetti D.
      • Viola M.
      • Karousou E.
      • De Luca G.
      • Passi A.
      Metabolic control of hyaluronan synthases.
      ,
      • Hascall V.C.
      • Wang A.
      • Tammi M.
      • Oikari S.
      • Tammi R.
      • Passi A.
      • Vigetti D.
      • Hanson R.W.
      • Hart G.W.
      The dynamic metabolism of hyaluronan regulates the cytosolic concentration of UDP-GlcNAc.
      • Wang A.
      • Sankaranarayanan N.V.
      • Yanagishita M.
      • Templeton D.M.
      • Desai U.R.
      • Sugahara K.
      • Wang C.P.
      • Hascall V.C.
      Heparin interaction with a receptor on hyperglycemic dividing cells prevents intracellular hyaluronan synthesis and autophagy responses in models of type 1 diabetes.
      ), is also inhibited by AMPK (
      • Vigetti D.
      • Clerici M.
      • Deleonibus S.
      • Karousou E.
      • Viola M.
      • Moretto P.
      • Heldin P.
      • Hascall V.C.
      • De Luca G.
      • Passi A.
      Hyaluronan synthesis is inhibited by adenosine monophosphate-activated protein kinase through the regulation of HAS2 activity in human aortic smooth muscle cells.
      ). This is due to the specific AMPK-evoked phosphorylation of hyaluronan synthase 2 at Thr110, a modification that blocks its enzymatic activity (
      • Vigetti D.
      • Clerici M.
      • Deleonibus S.
      • Karousou E.
      • Viola M.
      • Moretto P.
      • Heldin P.
      • Hascall V.C.
      • De Luca G.
      • Passi A.
      Hyaluronan synthesis is inhibited by adenosine monophosphate-activated protein kinase through the regulation of HAS2 activity in human aortic smooth muscle cells.
      ). Hence, along with the induction of autophagy, endorepellin may also simultaneously alter the cellular microenvironment to favor angiostasis by reducing the expression of this pro-angiogenic glycosaminoglycan. Given this information along with previous in vivo data depicting endorepellin as a powerful means to curtail tumor growth and angiogenesis (
      • Bix G.
      • Castello R.
      • Burrows M.
      • Zoeller J.J.
      • Weech M.
      • Iozzo R.A.
      • Cardi C.
      • Thakur M.L.
      • Barker C.A.
      • Camphausen K.
      • Iozzo R.V.
      Endorepellin in vivo: targeting the tumor vasculature and retarding cancer growth and metabolism.
      ), we believe that our findings from this study may yield unique therapeutic targets for novel drug design.
      We should point out, however, that, although compound C typically crosses the plasma membrane to inhibit AMPKα, it also has affinity for VEGFR2. Because of this interaction, compound C can actually inhibit neovascularization through down-regulation of VEGFR2 signaling in some models of angiogenesis (
      • Cannon J.E.
      • Upton P.D.
      • Smith J.C.
      • Morrell N.W.
      Intersegmental vessel formation in zebrafish: requirement for VEGF but not BMP signalling revealed by selective and non-selective BMP antagonists.
      ). Indeed, we saw a non-significant reduction in vessel number in rings treated with compound C alone compared with vehicle. As endorepellin binds and signals through VEGFR2 (
      • Goyal A.
      • Pal N.
      • Concannon M.
      • Paul M.
      • Doran M.
      • Poluzzi C.
      • Sekiguchi K.
      • Whitelock J.M.
      • Neill T.
      • Iozzo R.V.
      Endorepellin, the angiostatic module of perlecan, interacts with both the α2β1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2).
      ,
      • Goyal A.
      • Poluzzi C.
      • Willis C.D.
      • Smythies J.
      • Shellard A.
      • Neill T.
      • Iozzo R.V.
      Endorepellin affects angiogenesis by antagonizing diverse VEGFR2- evoked signaling pathways: transcriptional repression of HIF-1α and VEGFA and concurrent inhibition of NFAT1 activation.
      ), it is possible that compound C may interfere with the endorepellin/VEGFR2 axis.
      Remarkably, our findings are corroborated by other studies that depict a number of angiostatic matrix proteins and their domains, including endostatin (
      • Nguyen T.M.
      • Subramanian I.V.
      • Xiao X.
      • Ghosh G.
      • Nguyen P.
      • Kelekar A.
      • Ramakrishnan S.
      Endostatin induces autophagy in endothelial cells by modulating Beclin 1 and β-catenin levels.
      ) and Kringle V (
      • Nguyen T.M.
      • Subramanian I.V.
      • Kelekar A.
      • Ramakrishnan S.
      Kringle 5 of human plasminogen, an angiogenesis inhibitor, induces both autophagy and apoptotic death in endothelial cells.
      ), which can concurrently induce autophagy. Notably, a recent study also illustrated that the natural compound capsicodendrin exhibits angiostatic activity and autophagy induction via VEGFR2 inactivation (
      • Pan C.C.
      • Shah N.
      • Kumar S.
      • Wheeler S.E.
      • Cinti J.
      • Hoyt D.G.
      • Beattie C.E.
      • An M.
      • Mythreye K.
      • Rakotondraibe L.H.
      • Lee N.Y.
      Angiostatic actions of capsicodendrin through selective inhibition of VEGFR2-mediated AKT signaling and disregulated autophagy.
      ). Conversely, impairing autophagy in retinal epithelial cells leads to enhanced angiogenesis (
      • Liu J.
      • Copland D.A.
      • Theodoropoulou S.
      • Chiu H.A.
      • Barba M.D.
      • Mak K.W.
      • Mack M.
      • Nicholson L.B.
      • Dick A.D.
      Impairing autophagy in retinal pigment epithelium leads to inflammasome activation and enhanced macrophage-mediated angiogenesis.
      ). Thus, our study contributes new information in support of a paradigm shift whereby autophagy may act primarily as an anti-angiogenic mechanism.
      In summary, these studies have shown that both human and porcine endothelial cells undergo morphological and biochemical changes following treatment with endorepellin. In both cell types, we observed autophagosome formation in response to endorepellin treatment, accompanied by changes in cell surface roughness and height. Mechanistically, endorepellin-induced autophagy is dependent on AMPKα activation downstream of VEGFR2. This autophagic induction includes the activation of the pro-autophagic complex including Peg3, LC3, Beclin 1, and p62 (Fig. 9). This process is ultimately associated with down-regulation of mTOR signaling through autophagic clearance of this master inhibitor of autophagy as well as with reduced angiogenesis (Fig. 9). Future studies utilizing these properties of endorepellin may allow the development of new therapeutic modalities for treating devastating diseases, such as cancer, that involve aberrant angiogenesis.

      Experimental Procedures

      Cells and Materials

      Human umbilical vein endothelial cells (HUVEC) were grown in basal medium supplemented with the VascuLife EnGS Life Factors Kit (LifeLine Cell Technology, Frederick, MD), with cells being utilized within the first five passages. Porcine aortic endothelial cells expressing VEGFR2 (PAER2) were described previously (
      • Goyal A.
      • Pal N.
      • Concannon M.
      • Paul M.
      • Doran M.
      • Poluzzi C.
      • Sekiguchi K.
      • Whitelock J.M.
      • Neill T.
      • Iozzo R.V.
      Endorepellin, the angiostatic module of perlecan, interacts with both the α2β1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2).
      ,
      • Goyal A.
      • Poluzzi C.
      • Willis C.D.
      • Smythies J.
      • Shellard A.
      • Neill T.
      • Iozzo R.V.
      Endorepellin affects angiogenesis by antagonizing diverse VEGFR2- evoked signaling pathways: transcriptional repression of HIF-1α and VEGFA and concurrent inhibition of NFAT1 activation.
      ). The rabbit polyclonal LC3B antibody (L7543) was obtained from Sigma-Aldrich (St. Louis MO). The goat polyclonal LC3 antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, sc-16756). The rabbit primary antibodies against Beclin 1 (3738S), VEGFR2 (55B11), mTOR (7C10), AMPKα (2532), phospho-AMPKα at residue Thr172 (40H9), and GAPDH (14C10) were procured from Cell Signaling Technology (Danvers, MA). The rabbit CD31 (Ab28364) primary antibody was purchased from Abcam (Cambridge, MA). For immunoblots, all primary antibodies were used at a 1:1000 dilution, except GAPDH, which was used at 1:5000. Secondary antibodies were used at 1:4000 dilutions. For immunofluorescence studies, primary antibodies were used at a concentration of 1:200, and secondary antibodies were used at a 1:400 dilution. The goat anti-rabbit secondary antibody conjugated with HRP (12–348) was purchased from Millipore, Inc. (Billerica, MA). Donkey anti-rabbit Alexa Fluor 488 (A21206), donkey anti-rabbit Alexa Fluor 594 (A11012), and donkey anti-goat Alexa Fluor 594 (A11058) antibodies were purchased from Invitrogen. Compound C and gelatin were obtained from Sigma-Aldrich. SuperSignal West Pico chemiluminescence substrate was purchased from Thermo Fisher Scientific (Philadelphia, PA). RNAi targeting human VEGFR2 and corresponding control siRNA (siScrambled, denoted as siScr) were purchased from Santa Cruz Biotechnology. Lipofectamine RNAiMAX was acquired from Invitrogen. Human recombinant endorepellin was expressed and purified as described previously (
      • Goyal A.
      • Pal N.
      • Concannon M.
      • Paul M.
      • Doran M.
      • Poluzzi C.
      • Sekiguchi K.
      • Whitelock J.M.
      • Neill T.
      • Iozzo R.V.
      Endorepellin, the angiostatic module of perlecan, interacts with both the α2β1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2).
      ,
      • Goyal A.
      • Poluzzi C.
      • Willis C.D.
      • Smythies J.
      • Shellard A.
      • Neill T.
      • Iozzo R.V.
      Endorepellin affects angiogenesis by antagonizing diverse VEGFR2- evoked signaling pathways: transcriptional repression of HIF-1α and VEGFA and concurrent inhibition of NFAT1 activation.
      ,
      • Poluzzi C.
      • Casulli J.
      • Goyal A.
      • Mercer T.J.
      • Neill T.
      • Iozzo R.V.
      Endorepellin evokes autophagy in endothelial cells.
      ). Type I collagen was purchased from BD Biosciences. BS-1 Lectin was purchased from Thermo Fisher Scientific and Sigma-Aldrich. DAPI was purchased from Invitrogen.

      Scanning Cell Morphology and Ultrastructure by Atomic Force Microscopy

      A Dimension Icon atomic force microscope was used for nanostructural studies (BrukerNano, Santa Barbara, CA). A confluent monolayer of HUVEC or PAER2 cells was grown on a 0.2% gelatin-coated four-chamber slide (Thermo Fisher Scientific). After treatment, cells were washed in ice-cold PBS before fixing on ice for 2 h in glutaraldehyde diluted to 2% in HBSS. The fixed cells were washed again in PBS before drying in a desiccation chamber. Images were acquired in tapping mode using a nanosized silicon tip (NCHV-A; BrukerNano; tip radius R, ∼10 nm; spring constant, ∼42 N/m). To determine the elastic modulus of individual cells, cells were grown in either basal medium with growth factors ± 200 nm endorepellin (6 h) or HBSS (4 h) and tested without fixing. Assisted by an optical microscope to locate individual cells, nanoindentation was performed at 7 μm/s indentation depth rate using a microspherical tip (R ≈ 2.5 μm, k ≈ 0.1 N/m) and the Dimension Icon AFM in the same medium. A Hertz model with finite cell height correction (
      • Dimitriadis E.K.
      • Horkay F.
      • Maresca J.
      • Kachar B.
      • Chadwick R.S.
      Determination of elastic moduli of thin layers of soft material using the atomic force microscope.
      ) was applied to each force-depth loading curve to calculate the effective elastic indentation modulus. The Poisson's ratio of the cells was assumed to be 0.5 (
      • Nikolaev N.I.
      • Müller T.
      • Williams D.J.
      • Liu Y.
      Changes in the stiffness of human mesenchymal stem cells with the progress of cell death as measured by atomic force microscopy.
      ).

      Immunoblotting

      HUVEC were treated as necessary for the given analyses and lysed in radioimmune precipitation assay buffer (50 mm Tris (pH 7.4), 150 mm NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mm EDTA/EGTA/sodium orthovanadate, 10 mm β-glycerophosphate, and protease inhibitors (1 mm phenylmethylsulfonyl fluoride and 10 μg/ml leupeptin/tosylphenylalanyl chloromethyl ketone/aprotinin each)) for 20–25 min on ice. Resolved proteins were then transferred to nitrocellulose membranes (Bio-Rad), probed with the indicated antibodies, and developed with the enhanced chemiluminescence technique. Resulting chemiluminescent signatures were detected via an ImageQuant LAS-4000 (GE Healthcare) visualization platform as described previously (
      • Neill T.
      • Torres A.
      • Buraschi S.
      • Owens R.T.
      • Hoek J.B.
      • Baffa R.
      • Iozzo R.V.
      Decorin induces mitophagy in breast carcinoma cells via peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and mitostatin.
      ).

      Immunofluorescence Microscopy

      HUVEC, grown on 0.2% gelatin-coated four-chamber slides (Thermo Fisher Scientific), were treated for the respective analyses. Cells were subsequently washed with PBS and fixed on ice for 30 min in 4% paraformaldehyde at room temperature (
      • Rudnicka L.
      • Varga J.
      • Christiano A.M.
      • Iozzo R.V.
      • Jimenez S.A.
      • Uitto J.
      Elevated expression of type VII collagen in the skin of patients with systemic sclerosis.
      ,
      • Ryynänen M.
      • Ryynänen J.
      • Sollberg S.
      • Iozzo R.V.
      • Knowlton R.G.
      • Uitto J.
      Genetic linkage of type VII collagen (COL7A1) to dominant dystrophic epidermolysis bullosa in families with abnormal anchoring fibrils.
      ). Cells were blocked in PBS + 5% bovine serum albumin, incubated with various antibodies for 1 h, washed in PBS, and incubated for 1 h with an appropriate secondary antibody. Nuclei were stained and visualized with DAPI (Vector Laboratories). Fluorescence images were acquired with a ×63, 1.3 oil immersion objective using a Leica DM5500B microscope programed with the Leica Application Suite, Advanced Fluorescence v1.8, from Leica Microsystems, Inc. All resulting immunofluorescence images were analyzed using ImageJ software (National Institutes of Health) and Adobe Photoshop CS5.1 (Adobe Systems).

      Transient siRNA-mediated Knockdown

      Transient knockdown of VEGFR2 in HUVEC was achieved via transfection with validated siRNAs specific for VEGFR2 (sc-29318) from Santa Cruz Biotechnology. Scrambled siRNA (siScr, sc-37007) served as a control for all siRNA experiments presented here. Six-well plates were seeded with 2 × 105 HUVEC, followed by incubation at 37 °C + 5% CO2 until cultures were ∼70% confluent. Targeting or scrambled siRNA duplex was mixed with transfection medium and Lipofectamine RNAiMAX. After incubation at an ambient temperature (∼25 °C), the ribonucleic acid/cationic complexes were applied directly to the cells. Following a 48-h transfection, the cells were treated and lysed in radioimmune precipitation assay buffer. Verification of RNAi-mediated knockdown of the target protein was determined via immunoblotting.

      Aortic Ring Assays

      All animal protocols were performed according to the Guide for Care and Use of Laboratory Animals and the Institutional Animal Care and Use Committee of Thomas Jefferson University. Thoracic aortae from 5–7-week-old C57/BL6 mice (The Jackson Laboratory) were surgically isolated, cleaned, and dissected into 0.5-mm rings. Rings were embedded in 1 mg/ml of type I collagen in a 96-well plate as described previously (
      • Baker M.
      • Robinson S.D.
      • Lechertier T.
      • Barber P.R.
      • Tavora B.
      • D'Amico G.
      • Jones D.T.
      • Vojnovic B.
      • Hodivala-Dilke K.
      Use of the mouse aortic ring assay to study angiogenesis.
      ). When embedded, the rings were divided into respective groups: vehicle (PBS or DMSO), endorepellin (200 nm), Torin 1 (40 nm), compound C (1 μm), and compound C + endorepellin. Endothelial microvessel sprouts growing out from the rings were counted during the exponential growth phase to obtain angiogenic response data. Before the regression phase, rings were fixed for immunofluorescence staining of CD31 or BS-1 Lectin. Pictures were taken on day 12, and the total number of branches was counted using ImageJ.

      Quantification and Statistical Analysis

      Immunoblots were quantified by densitometry using ImageJ software. All experiments contained here were carried out with a minimum of three independent trials (
      • Iozzo R.V.
      • Chakrani F.
      • Perrotti D.
      • McQuillan D.J.
      • Skorski T.
      • Calabretta B.
      • Eichstetter I.
      Cooperative action of germline mutations in decorin and p53 accelerates lymphoma tumorigenesis.
      ). Results are expressed as the mean ± S.E. Statistical analysis was performed with SigmaStat for Windows version 3.10 (Systat Software, Inc., Port Richmond, CA). Significance of differences was determined by paired and unpaired Student's t test or one-way analysis of variance followed by Tukey-Kramer post-hoc multiple comparison. Data were considered significant with p < 0.05.

      Author Contributions

      R. V. I. conceived the study and coordinated the work. A. G., M. A. G., and R. V. I. performed experimental work and wrote the manuscript. D. R. C. performed the AFM measurements, data analysis, and interpretation. L. H. supervised the AFM work. All authors reviewed the results, contributed to data interpretation, and approved the final version of the manuscript.

      Acknowledgments

      We thank Lena Claeson-Welch for providing the porcine aortic endothelial cells overexpressing VEGFR2.

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